In Progress / Literature-based Diagnostics:
Serological: ELISA is available. Loi et al. (2002) developed monoclonal antibodies against apple proliferation phytoplasma. Brzin et al. (2003) showed that the ELISA procedure was very sensitive and reliable compared to PCR.
Grafting on sensitive indicator plants: Not recommended (very time intensive - up to 2 years). Malus x dawsoniana is a very sensitive indicator.
Green et al. (1999) developed an "easy and efficient" DNA extraction method from woody plants for detection of phytoplasmas by PCR.
Molecular: A "universal" PCR assay has been developed that enables amplification of the 16S rRNA genes of phytoplasmas. Digestion of these PCR products with selected restriction enzymes provides a DNA fingerprint in the form of 16S rDNA fragment patterns that can be used to determine phytoplasma identity (Deng and Hiruki, 1991; Ahrens and Seemuller, 1992; Lee et al., 1993; Smart et al., 1996; Gibb et al., 1999).
Nested PCR: Nested PCR has been employed for the detection of phytoplasmas both in plants and psyllids using universal primers (Deng and Hiruki, 1991; Gundersen and Lee, 1996) and/or 16SrX phytoplasma group specific primer pairs (Lee et al., 1995; Lorenz et al., 1995).
Seemuller and Schneider (2004) offer a summary of the molecular studies conducted on Ca. P. mali, Ca. P. prunorum, and Ca. P. pyri.
Immunocapture PCR (IC-PCR): Rajan and Clark (1995), use immunocapture-PCR to detect apple proliferation in apple bark. They used rabbit polyclonal antibodies to capture the phytoplasma and then amplified with universal PCR primers.
Real-time PCR: Jarausch et al. (2004) developed a quantitative real-time PCR for apple proliferation phytoplasma in plants and insects from a nitroreductase gene sequence. Galetto et al. (2005) developed an apple proliferation specific real-time PCR assay from the same nitroreductase gene sequence. These authors also developed a universal assay for detection of phytoplasmas belonging to groups 16Sr-V, 16Sr-X, and 16Sr-XII.
Torres et al. (2005) developed a real-time PCR that will detect Ca. P. mali, Ca. P. prunorum, and Ca. P. pyri (three phytoplasmas in apple proliferation group of quarantine importance).
Baric and Dalla-Via (2004) developed a real-time PCR for apple proliferation phytoplasma in apple plant material. The assay also amplified a host gene from apple as an internal control.
Baric et al. (2006) compared real-time PCR with four conventional PCR assays. The real-time procedure had the highest sensitivity and specificity and was not susceptible to PCR inhibition. The one downfall was the high cost of the procedure.
Aldaghi et al. (2007) developed a real-time PCR protocol for Ca. Phytoplasma mali. This probe could distinguish a single mismatch between Ca. P. mali and Ca. P. prunorum, but late fluorescent curves were obtained from European stone fruit isolates.
Aldaghi et al. (2008) developed a new probe and adapted the original procedure to eliminate the late fluorescent curves.